In 1981 human pancreatic growth hormone releasing hormone (hGH-RH) was first isolated from extracts of human pancreatic tumors and subsequently from the hypothalamus of various mammals. This peptide was found to promote the release of growth hormone (GH) by the pituitary. The human hypothalamic GH-RH was found to have the same amino acid sequence as the pancreatic one. Human GH-RH (hGH-RH) contains 44 amino acids with an amidated carboxyl terminus. The structure of hGH-RH was reproduced by synthesis. Several analogs of hGH-RH have been synthesized and their biological activity studied. These studies revealed that:
a) a fragment of GH-RH containing at least 29 amino acid residues has at least 50% of the potency of natural GH-RH; further deletion of amino acid residues results in a marked decrease in bioactivity [Cambell R M et al. Peptides 12: 569-574 (1991)];
b) replacement of Arg in position 29 by Agm (agmatine, 4-guanidino-butylamine) is said to provide resistance to enzymatic degradation from C-terminus (Bajusz S et al. in Peptides, 1982, Blaha and Melon, Eds., Walter de Gruyter, Berlin-N.Y., 1983, pp. 643-647);
c) replacement of Tyr in position 1 by des-aminotyrosine (Dat) is said to lead to analogs with increased biological activities as a result of the resistance of N-terminus to enzymatic degradation [Felix A et al. Int. J. Peptide Protein Res. 32: 441-454 (1988), Kovacs M et al. Life Sci. 42: 27-35 (1988)]. U.S. Pat. Nos. 4,622,312, 4,649,131 and 4,784,987 disclose hGH-RH(1-29) agonists with Ala at position 15, as well as Dat at position 1. Several of these agonists are said to have potency four-fold that of hGH-RH(1-29);
d) several analogs containing Dat in position 1 and Agm in position 29 are said to exhibit enhanced GH releasing ability, and hence potency greater than that of hGH-RH(1-29) and in vivo [Zarandi M et al. Int. J. Peptide Protein Res. 36: 499-505 (1990), Zarandi M et al. Int. J. Peptide Protein Res. 39: 211-217 (1992)];
e) hydrophobic groups at the C-terminal of a peptides sequence can result in significantly increased specific activity. In terms of hydrophobicity, these results are contradicted by works of other groups, e.g. Muranichi [Muranichi S et al. Pharm. Res. 8: 649-652 (1991)];
Similarly, U.S. Pat. No. 4,914,189 discloses agonists having Dat at position 1, D-Ala at position 2, Lys or Orn at position 12, Gly at position 15, Lys at position 21 and Agm at position 29. It should be noted however that those agonists said to have had greater potency in inducing GH release than hGH-RH(1-29) had Lys at position 12 and D-Ala at position 2.
In U.S. Pat. No. 4,689,318 analogs of hGH-RH(1-29) may have Lys12 or Lys21 replaced by Orn and in which position 8 could be Ser (rat GH-RH sequence), Asn (human GH-RH sequence), Thr (mouse GH-RH sequence) or Gln (not naturally occurring in GH-RH); and position 27 could be Nle. In these analogs, position 1 is never Dat, position 15 never Abu, position 28 never Asp, and position 29 never Agm, Arg-NH—CH3, or Arg-NH—CH2—CH3. Those analogs said to have potency as strong as hGH-RH(1-40) had substitutions by Arg at positions 12 and 21.
Other hGH-RH(1-29) agonists are taught in PCT patent applications numbers WO 94/11396 and 94/11397, where at position 12, Lys12 is to be replaced by D-Lys, Arg or Orn. These analogs may also contain Dat as R1; Asn, D-Asn, Ser, D-Ser as R8; Abu as R15; Lys, D-Lys, Arg or D-Arg as R21; Nle as R27; Asp or Ser as R28; and Agm as R29. Those agonists which are said to induce GH at levels exceeding those induced by hGH-RH(1-29) had Lys at positions 12. The teachings of these two publications however are considered open to question since, some time after the filing of these applications, it was discovered that the compounds believed to have been synthesized could not with certainty be said to correspond with the formulae they were originally paired with. Moreover, it was further discovered after filing these applications that the compounds could not release GH at the levels originally asserted.
European Patent Application 0 413 839 discloses further hGH-RH analogs in which positions 12 and 21 may both be Lys or Orn, and where position 15 is Ala. However, those analogs tested for GH releasing ability and said to have greater potency than hGH-RH(1-29) had Lys at positions 12 and 21.
U.S. Pat. No. 5,262,519 discloses agonists having the following substitutions: N-MeTyr at position 1, Ser at position 8, Ala at position 15, Ala or Aib at positions 16, 24, and 25, Asn at position 28, and —NHR at the C-terminus with R being H or lower alkyl. It should be noted however that these agonists said to have had greater potency in inducing GH release than hGH-RH(1-29) never had Gln or Thr at position 8, Orn at positions 12 and 21, Abu at position 15, and Asp at position 28.
U.S. Pat. No. 5,792,747 discloses GH-RH agonists having Dat at position 1; Glu at position 3; Ser, Gln, or Thr at position 8; Orn at positions 12 and 21; Ile at position 13; Ala or Abu at positions 15, 22, and 23; Glu at position 25; Nle, Ile, or Leu at position 27; Asn or Asp at position 28; and Agm at position 29. It should be noted however that those agonists said to have had greater potency in inducing GH release than hGH-RH(1-29) never had N-Me-Tyr1, D-Ala2, and/or —NH—CH3 or —NH—CH2—CH3 at the C-terminus.
U.S. Pat. No. 7,928,063 discloses GH-RH agonists having the following substitutions: Dat at position 1; 6-guanidino-2-caproic acid (hArg), 4-guanidine-2-aminobutyric acid (Gab), or 3-guanidino-2-aminopropionic acid (Gap) at positions 11 and 20; Orn, hArg, Gab, or Gap at positions 12 and 21; Ala at position 15, Nle at position 27; and D-Arg, hArg, Gab, or Gap at position 29. These peptides are said to exhibit high resistance to enzymatic actions and are potent and selective GH release stimulators. It should be noted however that these agonists have been tested and showed high stability only in trypsin digestion test.
Up to now, most of the GH-RH analogs described have been tested in rat models, either in vitro or in vivo. Since human and rat GRF(1-29)NH2 are markedly different, the structure-activity relationships of GH-RH are different in both species. Therefore, it is not possible to extrapolate results obtained in rats to humans. (Brazeau et al. U.S. Pat. No. 6,458,764).
Other hGH-RH(1-29) analogs are taught in US published application 2009/0023646 A1 and WO 2009/009727 A2, where the most potent compound has Ala2, Tyr10, Gly15, and Leu22 replaced by D-Ala2, D-Tyr10, D-Ala15, and Lys22, respectively, showed binding to GH-RH receptor on somatotrophs in rat and dog pituitaries and was at least two times more potent in vivo than the natural GH-RH(1-44).
An analog of hGH-RH(1-44)NH2 (tesamorelin) that was modified by trans-3-hexenoyl group at the N-terminus showed resistance against DPP-IV catalyzed deactivation [Ferninandi E S et al. Basic Clin Pharmacol Toxicol. 100: 49-58 (2007) and Falutz J et al. Acquir Immune Defic Syndr 53: 311-322, (2010)]. It should be mentioned however that this agonist was not protected against endopeptidases and chemical degradation in aqueous solution and was only about twice as active as GH-RH itself.
Native hGH-RH(1-44) and its analogs are subject to biological inactivation by both enzymatic and chemical routes. In plasma, hGH-RH is rapidly degraded via dipeptidylpeptidase IV (DPP-IV) cleavage between residues 2 and 3 [Frohman et al., J. Clin. Invest. 83, 1533-1540 (1989), Kubiak et al. Drug Met. Disp. 17, 393-397 (1989)]. the major cleavage site in plasma. In pituitary and hypothalamus, the major cleavage sites are between Leu14-Gly15 (chymotrypsin-like enzymes) and between Lys21-Leu22 (trypsin-like enzymes) [Boulanger et al. Brain Res. 616, 39-47 (1993)]. Other trypsin specific cleavages at basic amino acid residues are also observed. The hGH-RH(1-44) is also subject to chemical rearrangement [forming Asp8 or beta-Asp8 from Asn8 via aminosuccinimide formation] and oxidation [Met(O)27 from Met27] in aqueous environment that greatly reduce its bioactivity.
It is therefore advantageous to develop long-acting GH-RH analogues using specific amino acid replacements at the amino-terminus (to prevent enzymatic degradation), at residue 8 (to reduce isomerization), and residue 27 (to prevent oxidation). Inclusion of Ala15 or Abu15 substitutions for Gly 15, previously demonstrated to enhance receptor binding affinity, also improves GH-RH potency.
It would be desirable to produce hGH-RH analogs by multiple amino acid substitutions that have elevated binding affinities to the pituitary receptors in vitro, and increased potencies in vivo as compared to the native hGH-RH(1-29)NH2.
Since one change in the amino acid sequence of a peptide could cause a big change in the three dimensional structure of the peptide which has influence on the binding property as well as the biological potency of the peptide, it is impossible to predict which one or more amino acid replacements or combinations of substitutions in hGH-RH analogs might result in improved binding affinity or high in vivo potency.
Although the invention has been described with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto. For example, modifications in the peptide chain, particularly deletions beginning at the carboxyl terminus of the peptide and extending to about position-29, can be made in accordance with the known experimental practices to date to create peptides or peptide fragments that retain all or very substantial portions of the biological potency of the peptide, and such peptides are considered as being within the scope of the invention. Moreover, additions may be made to either terminus, or to both terminals, and/or generally equivalent residues can be substituted for naturally occurring residues, as is well-known in the overall art of peptide chemistry, to produce other analogs having at least a substantial portion of the potency of the claimed polypeptide without deviating from the scope of the invention. Moreover, modifications may be made to the preferred —NH2 group at the C-terminus in accordance with the state of this art today; for example, the carboxyl moiety of the amino acid residue at the C-terminus can be the radical —COOR, —CRO, —CONHNHR, —CON(R)(R′) or —CH2—OR, with R and R′ being lower alkyl, fluoro lower alkyl or hydrogen, without deviating from the invention, for such modifications result in equivalent synthetic peptides. (Rivier U.S. Pat. No. 5,262,519).